The long-range goal of the proposed studies is to identify the mechanisms of cell fate specification and gastrulation during vertebrate development. Our focus is on the Nodal TGFbeta signaling pathway, which plays fundamental roles during gastrulation and left-right axis determination in vertebrates. In particular, Nodal signals are concentration-dependent inducers of mesodermal and endodermal progenitors and required for cell internalization during gastrulation. The proposed research addresses three fundamental questions: (A) How are Nodal signals interpreted in time and space? (B) How is Nodal signaling regulated? (C) Which genes mediate the effects of Nodal signaling during gastrulation? To address these questions, the following three aims are proposed. (1) To determine how Nodal signals are interpreted, the expression of Nodal signals will be modulated and the distribution of the Nodal effector Smad2 will be visualized in live embryos. This aim will test the hypothesis that cells constantly monitor Nodal signals and chose their fate according to both signal concentration and exposure time. (2) To determine the role of microRNAs in the regulation of Nodal signaling, microRNA function will be altered and the phenotypic consequences analyzed. This aim will test the hypothesis that microRNAs maintain Nodal signaling at equilibrium by dampening the expression of Nodal signaling components. (3) Nodal-regulated genes have been identified that are involved in the internalization and differentiation of mesodermal and endodermal progenitors. To determine the role of Nodal-regulated genes during gastrulation, in vivo imaging and mutant analysis will be combined. Abnormalities in Nodal signaling components in humans are associated with forebrain anomalies, left-right defects and cancer. The proposed studies should thus help to provide the necessary context for understanding human birth defects and disease. The in vitro generation of endoderm or mesoderm is crucial to develop cell-based therapies for regenerative medicine. The proposed studies will thus help to inform strategies to induce stem cells to differentiate into organ progenitors.